Introduction Euphorbia pulcherrima 2004 2005 2005 1995 2002 2006 2006 2004 http://www.florigene.com 1997 2004 2006 2006 1997 2001 2006 1987 2004 1994 2001 2006 Agrobacterium Poinsettia mosaic virus 2000 Tymoviridae 2005 1980 2002 2005 Agrobacterium 1998 1998 2005 2004 2001 1999 2000 2001 2002 2000 2001 2003 1997 2004 2006 2006 A. tumefaciens Materials and methods Plant materials Euphorbia pulcherrima A. tumefaciens Somatic embryogenesis of poinsettia cv. Millenium 2 1994 1994 1962 −1 −1 −1 1 −1 −1 −1 1 −1 −1 1 −1 −1 −1 1 1994 1 Table 1 1994 Medium MS Sucrose (%) −1 −1 −1 −1 −1 CI Full strength 3 0.2 0.2 – – – SEI Full strength 3 – – 0.3 0.15 – SEM Full strength 3 – 0.05 – – – RI 1/2 strength 2 – – – – 2 HFMS 1/2 strength 2 – – – – – CI SEI SEM RI HFMS −2 −1 −2 −1 Agrobacterium A. tumefaciens 1983 1 Xho Kpn Cla Xba Xho EcoR Cla Xba 2 pdk 1 2003 1992 Not 2003 1 nptII 1 A. tumefaciens Fig. 1 a b 2 Materials and methods CP R2 R3  Table 2 Primers used in this study Primer name Restriction sites a Location R2F-XhK Xho Kpn ctcgagggtaccTTTAGCAAAACGCAGCACAAAATCA 4,371–4,395 R2R-CXb Cla Xba catcgattctagaTCTCCAGACACCATGATTGGGTG 4,848–4,870 R3F-XhK Xho Kpn ctcgagcggtaccTTCGCTTTAAAACAGAAAGCACCA 4,939–4,962 R3R-CXb Cla Xba catcgatgtctagaGCCTCGTAGCTTGGTTGGGTT 5,418–5,438 CPF-XhE Xho Eco ctcgaggaattcAACCACGTCGACTCCACTCCAT 5,478–5,499 CPR-CXb Cla Xba atcgattctagaAGCTTGCCGCTCACCAGCAC 5,958–5,977 a Transformation of poinsettia stem explants A tumefaciens −1 600 Agrobacterium Selection and regeneration of transgenic plants −1 −1 −1 −1 −1 −1 1 Screening of transgenic poinsettia plants  2 2 Southern blot analysis 1989 1988 Hind Hind Hind 32 1 Northern blot hybridization of low molecular weight RNA 4 1 1999 Virus inoculation and detection N. benthamiana 1977 3 3 3 p −1 A 450 Results Establishment of somatic embryogenesis for poinsettia cv. Millenium 3 2 3 2 2 2 2 3 Table 3 Experiments and efficiency of somatic embryogenesis Experiment No. of explants % of embryogenic explants Range of SE/embryogenic explant a b 1 200 21 2–10 5.3 ± 2.4 70 2 200 37 2–14 5.8 ± 3.1 135 3 350 75 3–18 7.2 ± 4.1 768 SE a b Fig. 2 a arrows bar b bar c d  The RI and HFMS media used for root induction did not show any clear differences in the percentage (85 and 87% for RI and HFMS, respectively) of plants that developed roots 3 weeks after being transferred to the rooting media. A. tumefaciens A. tumefaciens 4 4 Agrobacterium 4 Table 4 Agrobacterium Co-cultivation time (days) Inoculation time  5 min 10 min 20 min 2 9 (11%) 13 (16%) 21 (26%) 3 26 (33%) 21 (26%) 20 (25%) 6 10 (13%) 8 (10%) 3 (4%) Agrobacterium −1 −1 −1 −1 Production of transgenic poinsettia plants, molecular analyses and resistance assays 5 Table 5 Agrobacterium a No. of explants No. of regenerated plants b c 1 (CP) 185 54 3 1.6 2 (R3) 132 69 3 2.3 3 (R3) 172 66 6 3.5 4 (R2) 254 21 3 1.2 5 (R2) 125 18 3 2.4 a b c 3 5 Fig. 3 a) b) c) Lane A1 lane A2 lanes A3–5 lane A6 Lane B1 lane B2 lanes B3-8 lane B9 Lane C1 lane C2 lanes C3-11 lane C12 arrow  4 4 4 Fig. 4 Hin 1 Lane 1 lanes 2-4 lanes 5-6 lanes 7-9 lane 10  5 5 Fig. 5 a Lane 1 lanes 2–3 lanes 4–5 lane 6 b Lane 1 lanes 2 3 lanes 4 5 lane 6  6 6 Table 6 Immunological detection of PnMV on non-transformed and transformed poinsettia plants inoculated with PnMV Construct a b 6 wpi 10 wpi R2 72-1 0.1 0.1 72-2 0.1 0.2 R3 18-1 0.1 0.2 40B 0.8 2.4 41-2 0.5 2.6 62-1 0.1 0.2 41-5 0.7 2.8 CP 11-1 0.1 0.2 3-1 0.1 0.1 Control + 1 0.8 2.8 2 0.8 2.7 Control − 1 0.1 0.2 2 0.2 0.2 3 0.2 0.2 4 0.1 0.1 wpi a b Discussion Significance of genetic engineering in control of virus diseases in ornamentals 1994 1995 1997 1998 2000 2002 2004 2004 2006 1997 2006 gus 2002 Agrobacterium Agrobacterium 1991 1994 1999 2006 Euphorbia 3 1994 3 3 A. tumefaciens A. tumefaciens 4 1999 2002 2003 2004 2004 A. tumefaciens 2002 Agrobacterium 1996 1997 2001 Agrobacterium nptII −1 2002 2004 2005 −1 Agrobacterium Platanus acerifolia −1 2007 Agrobacterium 5 4 4 2004 2006 1988 1992 2000 2000 2007 2007 Agrobacetrium The hp RNA constructs and PnMV resistance in transgenic poinsettia cv. Millenium  2003 1986 1998 1998 2000 2002 2003 2000 Agrobacterium 6 5 6 Poinsettia is a non-food, non-feed and vegetatively propagated ornamental plant. Pollen mediated transgene outflow is restricted, a significant advantage over sexually propagated plants. The combination of genetic engineering and RNA silencing technologies for the generation of virus-resistant transgenic ornamentals is therefore appealing, especially in an industry in which quality is so important. Transgenic vegetatively propagated ornamentals like PnMV resistant transgenic poinsettia are likely to be more acceptable even in areas where genetically modified crops are currently not cultivated. A. tumefaciens 1997